CD44 is a polymorphic family of membrane glycoproteins expressed by many different kinds of cells. CD44 is found, for example, on circulating lymphocytes where it acts as a lymph node homing receptor, and on epithelial cells where it is involved with cell-cell adhesion (1). CD44 is also found in the lungs, on basal cells of the upper bronchi, pulmonary macrophages, alveolar lymphocytes and activated type II alveolar pneuomocytes, where it again functions in cell-cell adhesion (2). CD44 has a high affinity binding site for hyaluronan receptors, and CD44-hyaluronan interactions are used to anchor the cells in place (3).
Within a single cell CD44 may be expressed as two or more variants, presumably depending on the changing needs of the cell. The polymorphic diversity of the variants is generated by alternative splicing of the CD44 mRNA, which occurs when the various coding sequences (exons) of CD44 genes are transcribed (expressed) in different combinations. Protein products of splice variants (isoforms) of CD44 vary widely in size (from 110 kDa to more than 250 kDa) and in function. The extracellular domain of CD44 protein is known to involve cell-cell adhesion and the binding of extracellular matrix components including hyaluronic acid, fibronectin and collagen, while the intracellular domain of CD44 has been associated with ankyrin cytoskeletal proteins critical for CD44-dependent cellular mobility. CD44 has also been found to function in hematopoiesis and in lymphocyte infiltration into general circulation (4, 5, 6). The various functions of CD44 are intriguing because they can be used to explain similar behaviors between activated lymphocytes and metastasizing tumor cells. Both types of cells have relatively high expression of CD44, and both show invasive behavior, cell migration involving reversible adhesive contacts, accumulation and expansion in lymphoid tissue, and release into general circulation (6). The association with lymphoid tissue is especially interesting in that both lymphocytes and metastasizing tumor cells use CD44 variants to bind a specific ligand residing either in the extracellular matrix of the lymph nodes or on the surface of dendritic or other cells of the lymphoid tissue. Moreover, following growth and differentiation in the lymph nodes, both lymphocytes and tumor cells are synchronously released into the efferent lymphatic vessels in the general circulation. The release process requires a complex series of interactions between the lymphocytes and tumor cells, the extracellular matrix component and surrounding cells, and probably involves adhesion receptors, proteolytic enzymes, growth factors and growth factor receptors. These processes may be dependent upon clipping of the CD44 molecules, and specificity in the process may be mediated by tissue-specific ligands interacting with CD44 isoforms. Expression of CD44 in malignant cells is therefore an important factor in primary tumor growth, local invasiveness and metastatic proclivity (7,8,9).
The CD44 gene locus in human genome is on chromosome 11p13 (10). Recently, most of the genomic structure of the human CD44 gene has been established (11). Over a length of about 60 kilobases (kb), at least 20 exons are distributed. Ten of these encode sequences for the standard form of CD44 (exons 1-5 and 16-20). Between exons 5 and 16, at least ten further exons are localized, which are subjected to alternative splicing (exons 6-15). In humans as in other species, the CD44 gene codes a variety of alternatively spliced proteins having different sizes and functions. Several CD44 isoforms have been purified and characterized to date, including an 89-90 kDa glycoprotein referred to as the "standard" or "hematopoietic" isoform (CD44s), and 180 kDa or more glycoproteins referred to as "epithelial" or variant isoforms (CD44v). Isolation and characterization of cDNA clones encoding the standard and epithelial isoforms have shown that the protein sequences are identical except that the epithelial isoforms contain additional sequences of 134 or more amino acids arising from at least ten exons (v1-v10 ) which code for extra-cellular domain, and the epithelial isoforms are more heavily glycosylated (12, 29). While it is now recognized that the CD44 standard form plays a key role in the control of cell migration, the precise functions of the alternatively spliced CD44 variants, which predominate in most cell types are unknown.
The literature is not conclusive on the issue of whether CD44 expression, either in standard or variant forms, can be generally associated with metastatic potential in human tumors. A 1993 article, for example, points out that such a correlation has never been established (13). On the other hand, it is known that cells which express the highest levels of CD44 variant isoforms tend to be the cells that most often undergo malignant transformation. The uncontrolled growth of these cells coupled with the expression of CD44v isoforms and possibly other adhesion molecules might render them more invasive and metastatic. Also, numerous studies have found associations between malignant transformation or cancer metastasis and the expression of CD44 variant isoforms. For example, a 1994 article gives evidence that certain CD44 variants may help mediate human glioma cell adhesion and invasion (14,15). It has also been shown that uterine cervical carcinomas show strong expression of epitopes encoded by exons v7 and v8, which have not been detected in normal cervical epithelium (16). In another study, specific CD44 variants were shown to be over-expressed at particular stages of colorectal tumor progression, and an unfavorable prognosis has been suggested for colorectal tumors expressing CD44v isoforms previously associated with tumor metastasis (17, 18).
In some studies over-expression of only particular variants has been associated with cancers. For example, expression of variants containing exon v6 sequences occurs in the advanced stages of the development of some tumors, but some CD44 variants without exon v6 sequences appear at the earliest stage of tumorigenesis and in early adenomas. Screening of gastric adenocarcinoma has revealed CD44v expression in all tested specimens, with intestinal type adenocarcinomas expressing variant exons v5 and v6, and diffuse-type adenocarcinomas predominantly expressing only exon v5. Normal stomach mucosa has shown exon v5 expression within the foveolar proliferation zone and on mucoid surface epithelium (19). The same pattern of expression has been confirmed for exon v11 abundantly present in well differentiated intestinal tumors in comparison with diffuse type cancer tissues. Normal gastric mucosas have demonstrated significantly lower expression of exon v11 splice forms . CD44 presence has been associated with tumor recurrence and increased mortality during follow-up averaging 14 months.
There is indication in the literature that over-expression of CD44 has long term clinical significance. In one group of patients with positive expression of exon v6 in 87% of primary breast tumors and 100% auxiliary lymph node metastasis, poor overall survival correlated with the presence of CD44v epitopes (20, 21). Another group found similar results, but suggested that the effect diminishes with time. In a study on gliomas, invasiveness was highly inhibited in two cell lines and completely arrested in five other cell lines by a CD44-specific antisense oligonucleotide which inhibited CD44 expression (15).
To our knowledge, CD44 antisense therapy has never been applied against human lung cancer or melanoma, and it as never even been established that one or more variants of CD44 are associated with either of these types of neoplasm. The problem is exacerbated with respect to lung cancer because there are at least two major types, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). SCLC comprises approximately one-fourth of the cases, expresses neuroendocrine markers, and generally metastasizes early to lymph nodes, brain, bones, lung and liver. NSCLC comprises the majority of the remaining lung tumor types, and includes adeno-carcinoma, squamous cell carcinoma, and large cell carcinoma. NSCLC is characterized by epithelial-like growth factors and receptors, and is locally invasive. In 1994 Penno and colleagues reported that transcription and translation of CD44 standard form is consistently high among non-small cell tumors and squamous metaplasia of the lung, but is rare in small cell tumors and resting type II pneumocytes (2).
Melanocytes and melanoma cells have been widely studied as a model to investigate changes involved in malignant transformation, including the search for genes expressed in malignant, but not normal, melanocytes. Melanoma cells, unlike normal melanocytes, can proliferate in the absence of exogenous growth factors. This independence apparently reflects the production of growth factor and cytokines for autocrine growth stimulation (22).
Melanoma cells secrete a variety of growth factors including TGF-.alpha., TGF-.beta., platelet-derived growth factor A and B chains, basic fibroblast growth factor, IL-6, IL-1, granulocyte macrophage colony stimulating factor, and MGSA. These growth factors, expressed either constitutively or subsequent to induction with various cytokines, may contribute to the development of the transformed melanoma phenotype either by acting as autocrine growth factors or by modulating host response to the tumor cells (23, 24).
To produce metastasis, melanoma cells must detach from the primary tumor, invade through host stroma to gain entrance into the circulation, disseminate via the blood stream, and survive to reach distant capillary beds where they must attach, extravasate into the organ parenchyme, and proliferate into secondary growths. The growth of cells in distant sites occurs when the tumor cells produce autocrine growth factors or when the tumor cells respond to paracrine growth factors produced by host cells (22).
Approximately 95% of familial malignant melanomas and 40% of sporadic melanomas arise from precursor lesions. There are three types of melanocytic lesions: congenital, common acquired and dysplastic nevi. Congenital and common acquired nevi represent focal proliferations of normal melanocytes. In contrast, dysplastic nevi consist of a heterogenous population of normal melanocytes and melanocytes showing increased pigmentation, nuclear pleomorphism and mitotic atypia. For this reason, melanocytes of dysplastic nevi are considered to represent precursor lesions of human malignant melanoma. Human melanoma can be classified into three stages: i) melanoma in the radial growth phase; ii) melanoma in the vertical growth phase; and iii) metastatic melanoma. Primary melanoma in the radial and vertical growth phase and metastatic melanomas demonstrate significant biological, biochemical and karyotypic differences from normal human melanocytes and from melanocytic precursor lesions (23).
Human melanoma provides a well-suited model system because cells isolated from different stages of tumor progression can be cultured and studied in the laboratory. The human melanoma Hs294T (ATCC) used in the experiments discussed below was established from lymph node metastasis, and is a highly metastatic melanoma cell line (25). Taken in conjunction with the NSCLC experiments, the Hs294T experiments are thought to be predictive of the general effectiveness of the claimed therapies on neoplasms which over-express CD44.